1. Field of the Invention
The present invention relates to a microscope, specifically to an illuminating optical system that illuminates a sample using total reflection, and a fluorescence microscope provided with the same illuminating optical system. Also, the present invention relates to a microscope to which both of the conventional illumination using a reflecting illuminating optical system and the illumination using total reflection are applicable.
2. Description of Related Art
In recent years, in biological study, illumination method using total reflection (hereafter, referred to “evanescent illumination”) has been often employed for exciting fluorescent dye. The reason is as follows. According to this method, since the illumination range is extremely shallow in the depth direction of the sample, information on the surface and its vicinity of the sample is obtained at high sensitivity as being little affected by the background.
Comparison between the conventional reflecting illuminating optical system and the evanescent illumination is made as follows. As shown in FIG. 1, under the conventional reflecting illumination, illumination light is incident on a sample (not shown) on a cover glass via an objective lens along an optical axis. In this situation, illumination intensity is highest in the vicinity of the focal position of the objective lens and decreases as the position is away from the focal position. In this way, the illumination light has intensity distribution such that intensity varies in the depth direction of the sample. However, since the light has substantial intensity in a certain range, most of fluorescent dyes residing in the depth direction corresponding to this range are excited. Therefore, fluorescence emission other than from the surface in focus of the objective lens is observed as background light, to degrade S/N ratio.
On the other hand, as shown in FIG. 2, under the evanescent illumination, illumination light is incident on a sample via an objective lens, in an oblique direction in reference to an optical axis. In this situation, the irradiation angle is preset so that total reflection occurs at the interface between the cover glass and the sample. It is noted that not all the illumination light is reflected at the interface, but a very small part of the light oozes toward the sample side through the cover glass. The oozing light from the interface is evanescent light and reaches, in the depth direction of the sample, a distance equivalent to the wavelength of the light source in use. In this way, in the case where the evanescent light is used as illumination light, since the illumination range is limited to the depth equivalent to the wavelength of the light source in use, the region that emits fluorescence is very narrow while the remaining regions emitting no fluorescence, and accordingly the background fluorescence is very small, which is different from the case where the ordinary reflecting illumination is adopted. Therefore, evanescent illumination can achieve extremely high S/N ratio, and thus is specifically effective for observation of a cell membrane surface and for visualization of a single molecule of fluorescent dye that is localized in the vicinity of the cover glass surface.
Japanese Patent Application Preliminary Publication (KOKAI) No. Hei 9-159922, Japanese Patent Application Preliminary Publication (KOKAI) No. Hei 11-211990, and “Noninvasive Techniques in Cell Biology”, pp 93–127, Wiley-liss Inc. 1990, are known as disclosing the measures for embodying the evanescent illumination.
However, although the configuration disclosed in the above-mentioned prior art realizes evanescent illumination, laser light as the illumination light (exciting light) is designed to be introduced to a microscope via optical elements such as a reflecting mirror disposed between the laser light source and the microscope. Such a configuration requires adjustment of the optical elements, and, in addition, a cover for shielding the optical elements from dust, to render the apparatus bulky. Also, layout flexibility of the laser light source and the microscope is poor.
Also, in order to carry out the evanescent illumination, the irradiation angle of the illumination light on the sample is important. It is necessary to set the angle such that total reflection occurs. However, since the total reflection phenomenon is susceptible to deviation from the angle that causes total reflection, even a slight error of the irradiation angle induces failure of total reflection. One of causes of the angular error is vibration applied from outside. When vibration is conducted to the laser light source, the microscope and the optical elements, these members vibrate. As a result, irradiation position and the irradiation angle are subtly changed to cause error. Therefore, it is necessary to arrange the laser light source and the microscope integrally on an anti-vibration pedestal. In addition, it is necessary to provide, in the path for introducing laser light from the laser light source through the microscope, optical systems such as a beam expander, which expands the beam diameter of the laser light, and a collector lens. Since these optical systems also have to be arranged on the same anti-vibration pedestal, the resulting entire system becomes bulky.
Also, “Noninvasive Techniques in Cell Biology” describes that evanescent illumination is achieved with a mercury lamp and a conical prism having an annular opening (axicon prism) inserted in the illumination path. Use of an axicon prism certainly allows light that emanates from a single spot of a mercury lamp to be annularly shaped. However, since the luminous region of an actual mercury lamp is greater than 0.5 mm, annular beams of rays overlap each other, to fail to be a single annular beam of rays. Therefore, in the case of this method, it is necessary to pick up only a beam of rays emanating from a single spot of the mercury lamp by using a plurality of stops. However, since most of light emanating from the mercury lamp is cut off at the stops, the illumination intensity would be lowered.